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单细胞测序试验设计赏析（一）

单细胞测序试验设计中，单细胞测序技术通常会结合其它的技术来共同说明问题，或者结合年龄、性别等临床数据，进行分层分析说明问题以下以发表文章来进行一定的分析。

Single-cell RNA sequencing reveals the altered innate immunity in
immune checkpoint inhibitor-related myocarditis

PMID: 38425094 DOI: 10.1111/imm.13770
Abstract

Myocarditis has emerged as a rare but lethal immune checkpoint
inhibitor (ICI)-associated toxicity. However, the exact mechanism and the specific therapeutic targets remain underexplored. In this study, we aim to characterise the transcriptomic profiles based on single-cell RNA sequencing from ICI-related myocarditis. Peripheral blood mononuclear cell (PBMC) samples were collected from four groups for single-cell RNA sequencing: (1) patients with newly diagnosed lung squamous cell carcinoma before treatment (Control Group); (2) patients with lung squamous cell carcinoma with PD-1 inhibitor therapy who did not develop myocarditis (PD-1 Group); (3) patients during fulminant ICI-related myocarditis onset (Myocarditis Group); and (4) Patients with fulminant ICI-related myocarditis during disease remission (Recovery Group). Subcluster determination, functional analysis, single-cell trajectory and cell-cell interaction analysis were performed after scRNA-seq. Bulk-RNA sequencing was performed for further validation. Our results revealed the diversity of cellular populations in ICI-related myocarditis, marked by their distinct transcriptional profiles and biological functions. Monocytes, NKs as well as B cells contribute to the regulation of innate immunity and inflammation in ICI-related myocarditis. With integrated analysis of scRNA-seq and bulk sequencing, we identified S100A protein family as a potential serum marker for ICI-related myocarditis. Our study has created a cell atlas of PBMC during ICI-related myocarditis, which would shed light on the pathophysiological mechanism and potential therapeutic targets of ICI-related myocarditis in continuous exploration.

该单细胞测序的试验设计具有以下优点和一些可进一步考虑的方面：

优点：

• 合理的分组设置：试验设置了四个不同的组，包括未治疗的肺癌患者作为对照组、接受 PD-1 抑制剂治疗但未发生心肌炎的患者组、暴发性 ICI 相关心肌炎发作期患者组以及疾病缓解期患者组。这种分组方式能够全面地比较不同状态下外周血单个核细胞（PBMC）的转录组特征，有助于深入了解 ICI 相关心肌炎发生发展过程中细胞和分子层面的变化。
• 验证方法的应用：除了单细胞测序，还进行了大量 RNA 测序（Bulk-RNA sequencing）用于进一步验证。这种多组学结合的方法增加了研究结果的可靠性，通过不同测序技术的相互验证，能够更准确地确定与 ICI 相关心肌炎相关的分子特征和潜在标志物。

不同阶段，不同临床表型的单细胞测序从不同方面揭示疾病细胞和分子层面的变化，是试验设计中值得借鉴的；使用整体RNA测序进行验证也是常见的操作。

进一步考虑的方面：

• 缺乏其他样本类型：研究仅针对 PBMC 进行单细胞测序，而心肌炎是心肌组织的病变，可能还需要考虑对心肌组织进行单细胞测序，以更直接地了解心肌细胞在 ICI 相关心肌炎中的变化，进一步完善对疾病机制的理解。
• 未考虑其他影响因素：在研究设计中，未提及是否考虑了其他可能影响 PBMC 转录组的因素，如患者的年龄、性别、合并症等。这些因素可能对研究结果产生干扰，在后续研究中可以考虑对这些因素进行分层分析，以更准确地评估 ICI 相关心肌炎与细胞转录组变化之间的关系。